Children in developed countries are customarily protected against diphtheria using "DPT" vaccine a mixture of immunogens which induce the production of antibodies against diphtheria, whooping cough, and tetanus. Recently problems have arisen with respect to maintaining adequate supplies of this vaccine, apparently due to unwillingness of certain manufacturers to continue manufacturing. The reason for this reluctance is due to liability exposure in connection with lawsuits brought by parents of children having adverse reactions to the vaccine. At least some of these adverse reactions are assumed to be due to impurities in the vaccines.
The diphtheria component of the DPT vaccine 1s a toxoid produced by treating diphtheria toxin obtained from Corynebacterium diphtheriae, its natural source, with denaturants that permit its immunogenicity to be retained, while inactivating its cytotoxic properties. As this preparation involves the isolation of protein from natural sources and treatment of the mixture with nonspecific reagents, such as glutaraldehyde, a heterogeneous material ordinarily results. This presents a quality assurance problem which, evidently, has not yet entirely been solved.
Recombinant techniques offer a useful alternative to isolation of the native material. These techniques can be used to produce desired proteins which are free of those impurities that would ordinarily accompany the materials obtained from natural sources. In addition, recombinant techniques permit manipulation of the sequence of amino acids so as to confer desired properties on the product. Both of these advantages of recombinant protein production have been found relevant to the production of a superior diphtheria vaccine.
The entire coding sequence of diphtheria toxin (DT) has been cloned, and portions of it have been expressed in E. coli in practical amounts (Greenfield, L., et al, Proc Natl Acad Sci (1983) 80:6853-6857; Ratti, G., et al, Nucleic Acids Res (1983) 11:6589; Leong, D., et al, Science (1983) 220:515; Tweten, R. K., et al J Bacteriol (1983) 156:680).
The general characteristics of the toxin are well understood. DT is a typical toxin having an "A" fragment which is cytotoxic and "B" fragment responsible for its binding, and, possibly, translocation properties. DT is secreted from Corynebacterium diphtheriae as a single polypeptide chain containing 535residues. The approximately 193 residue sequence at the amino terminus is considered the A fragment; it is cytotoxic, because cf its enzymatic activity in catalyzing the transfer of ADP-ribose from NAD to elongation factor-2 (EF-2) thereby blocking protein synthesis and causing cell death. The carboxy-terminal, approximately 342 residue, sequence is considered the B portion and is not only associated with binding the cytotoxic portion to the cell but also presumably contains functions which permit translocation of the A fragment into the intracellular environment. The entire amino acid sequence of the naturally occurring toxin has been deduced from the coding sequence, and is set forth in FIG. 1. It is believed that cleavage after one of the Arg residues at positions 190, 192 or 193 (trypsin is capable of thus cleaving in vitro) is required for intoxication of cells. This alteration is believed to be followed by breakage of the disulfide bond between cysteine at position 186 and the cysteine at position 201 to liberate the A and B fragments from each other. Once released inside the cell, a single molecule of fragment A can be lethal for the individual cell (Yamaizumi, M., et al, Cell (1978) 15:245).
It is believed that the hydrophobic sequence of approximately 32 amino acids in the vicinity of residue 350 is responsible for the translocation properties of B chain. A cross-reacting mutant secretes "CRM 45", a modified form of DT which contains approximately half of the B portion at the N-terminal end. CRM45 is capable of insertion into artificial lipid bilayers under appropriate in vitro conditions to form ion conductive channels (Kagan, B. L., et al, Proc Natl Acad Sci (USA) (1981) 4950; Donovan, J. J., et al, ibid (1981) 78:172 Kayser, B., et al Biochem Biophys Res Commun (1981) 99:358). In addition, the 25 (32) amino acid region referred to above was earlier identified by Lambott, P., et al, J Cell Biol (1980) 8:837 as especially hydrophobic, and to resemble transverse lipid-associating domains found in intrinsic membrane proteins.
Other cross-reacting mutants have been disclosed. One of them, CRM197, is capable of binding target cells, but does not kill them. CRM197 has been shown to be enzymatically inactive, and to be nontoxic to guinea pigs (Uchida, T., et al, J Biol Chem (1973) 248:3838-3844). The gene encoding CRM197 has been sequenced, and shown to possess a single mutation in its A portion (Giannini, G., et al, Nucleic Acids Res (1984) 2:4063-4069).
It has also been shown recently that the glutamic acid residue at position 148 is critical to the active site (Caroll S. F., et al, Proc Natl Acad Sci (USA) (1984) 81:3307-3311). When NAD.sup.+ is chemically linked to glu-148 of native diphtheria toxin A fragment, enzymatic activity is lost. Inactivation of the A portion of DT offers the opportunity to provide a non-toxic protein which retains the immunogenicity of the native material.
The disadvantages of the currently available, relatively impure toxoid-based diphtheria component of the DPT vaccine could be overcome by the use of recombinant protein. The current toxoid preparations require stringent monitoring for toxicity, and, because glutaraldehyde linkage is random, are heterogeneous products. The recombinant vaccine provides increased yields of pure product without the necessity of time-course toxicity tests, due to the opportunity to modify specifically the toxic component to destroy its enzyme activity. The inactive A fragment is thus available as a homogenous preparation.